MXPA97009981A - Control of pests of hemipheral insects with bacillus thuringien - Google Patents

Abstract

The present invention relates to the use of endotoxins of Bacillus thuringiensis (B.t.) to control hemipteran insect pests, specifically exemplified by strain 201 T6 of B.t., B.t. var. israeleusis and the delta-endotoxins thereof, these isolates and toxins can be administered to said pests, or the environment of said pests, for example, plants, to achieve the control of the pests, a preferred embodiment is the transformation of the plants to express B toxins

Description

CONTROL OF HEMIPHERAL INSECTS PLfiGFIs WITH BfiCILLUS THURINGIENSIS RECIPROCAL REFERENCE fi UNfi RELIfiFIED APPLICATION 5 This application is a continuation that forms part of the co-pending application Sene Mo. 08/475, 97 filed on 07 Jumo de L (:) 95.

CfiTY OF LEF INVENTION The present invention relates to methods for controlling insect pests of Heiniptera order. In particular, it has been unexpectedly discovered that 6-endotox? ñas lb de liaeiilus t hurí nqierts. s (.B.t.) control beaches of heinius insects, for example, Lygus hespe us.

BACKGROUND OF THE INVENTION 0 The soil microbial Bacillus thuringiens s (B.t.) is a positive spore-forming bacterium Grarn characterized by parasporal inclusions of crystalline phase. Frequently, these inclusions appear microscopically as crystals of different shapes. The proteins can be highly toxic to pests and specific in their toxic activity. Certain genes for toxin (Je Bt.) Have been isolated and their sequence has been determined, and recombinant HDN-based products have been produced and approved for use in addition, with the use of genetic engineering techniques, New procedures for releasing Bt endotoxins into aggregative environments are under development, including the use of genetically engineered plants with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as vehicles for the release of 10 Bt endoto ina (Gaertner, FH, L. Kim C19B0 J TTBTECH 6. 4"- < .7). Rsi, the genes for endo-to ina of Bt. isolates are becoming eorneroi to inont e val Bears "Until the last fifteen years, the commercial use of Bt ,, pesticides has been largely restricted to a range narrow of lepidopteran pests (caterpillars). Preparations of The spores and crystals of B. t h u r i n g i e n i s v *? i *. ur tal-- 1 have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thur ngiensi var .. t - ur tal--? HD-l produces 6-endotox crystals? 20 that are toxic 20? ar * to the larvae of numerous lepid pterids. In recent years, however, researchers have discovered B.t. pesticides. with specific characteristics for a much wider range of pests. For example, other species of B.t. , namely *, B.t. var. israelensis '.r > and B. var. tenebrioni s (aa n ~ 7, aa Bt var di san di go), have been used commercially to control the sects of the Díptera and Coleóptera orders, respectively (Gaertner, FH ri989) "Cellular- Del ivery ysterns for * Insect icidaL Protems: Living and Non-Livmg i Croorganisms, "in Contled Delivery of Crop Protection flgents, RM Uilkins, ed., Taylor and Fr * anc? S, New York and London, 1990, pp. 245 255). See also Couch, T.L. (1980) "Mosquito Pathogenicity of Bacillus t hurí ng ensi var. I sraelen is," Developments in Industrial llicrobiology 22: b 1-76; and Beegle, 0. C. (1970) "Use of Kntornogenous Bacteria in figroecosystems", "Developme t m Industr lal Microbiology 21) .9" '-104 Kr'iey et al., Kr'ieg, A., A.

M »Huger, ü.A. Langenbruch, UJ. Schnetter I "1983] Z., ng. Ent" 9f * .500-500), describes Bacillus thuri ng ensí s var teneb ionis, which has been reported to be active against two beetles in the Coleoptera order. of the potato of Colorado Lep ri note rsa decernlineata and the beetle Age l as t ica to L ni. Recently, new subspecies of B. t "have been identified, and genes responsible for the active proteins of endotoxma (Hofte, H., H, R. Whiteley f 1 <3891 M crobiological Rev 52 (2) .242-255) Hofte and Uhiteley classified genes for crystal protein from B. t "in 4 major classes. classes were CryT (specifies epidopt er * a), C yl I (specifies pidoptera and Diptera), Cr-yTTT (specifies Coleoptera) and CryTV (specifies Diptera) Pre on aine and others (Prefontai ne, G ., P. Fast, PCK Lau, M.A. Hefford, Z. Hanna, R. Brosseau [19871 Appl. They sent me.

Microbe! . 5JÍ): 808-2814 J described probes used to classify active genes against lepioptera. It has been reported the discovery of strains that are toxic to other pests (Kei toison, J., 3. Payne, L. Knn I "1992.1 Bio / Technology 10: ??? 275.) The toxins Cr *? stal irias of Bt. are r * econocen geno or mind for being * protox nas, requirriendo physicochemical conditions part i «* -ul e < - (is docii ', pH, oxide reduction, ionic resistance ) or the activation of an active toxin (llofte and Whiteley, cited above), or both, in most cases, or both. insect provides conditions for the toxin to accumulate; However, cases have been documented where the prisoner 1 located or prepro eol i if they have been necessary for an optimal activity (Jacquet, 3., Huttor, P, l uthy l "19Bp" Speci dc ty of Bací 11 us t hurí ngien í s of t -ondotoxi n "Appl., Frivi ron.

My c! . 53: 500 - 5U4 io activity detection (Lainbert, fl., HR Holte, K. Annys, S. Jansens, P. Soetaert, M. Peferoen F19923"Novel Bací 11 us t huringí ensí s insect ic Lúa 1 cr- ystaL? r * otem with a silent activity against coleopteran lar * vae ", Appl. They sent me. Microbiol. 58: 2536-2542. The cloning and expression of a gene for a crystalline lens of B "t" in P cheri chía coLi has been described in the published literature (chnepf, H: E ", H..R .. Uhiteley [19811 Proc. Nati, Acad. Sci .. UA 78: 2093-2897). The Patent of E.U.A. 4,448,885 and the Patent of F.U.A. ¿. , 46, 036 describe the expression of crystal proteins of B.t. jan . 11. The Patents of E.U.A. 4,797,276 and 4,053 / 331 describe jL t hurí ngiensis var. tenebponis (.1-. a. B.t. san diego, a. k.

M-r7) that can be used to control * coleopteran pests in various environments. The Patent of E.U.A. 4,918,006 describes toxins of Bacillus thuringiensis var *. They are active against diptera pests, and report that a protein of approximately 27 kDa, and fragments thereof, are responsible for the activity against diptera. The Patent of E.U.A ,. No. 4,849.21 i7 describes isolates of li.t "having activity against the alphaLfa weevil. The Patent of E.U.A. No. 5,151,363 and the Patent of F.U.A. No. 4,948,734 describe certain isolates of B.t. that have activity against nematodes.

As a result of extensive research and investment of resources, other patents have been issued for new isolates of B "t and new uses of B.t. Isolates, however, the discovery of new isolates of B. t" and new uses of ai known sides of B.t, continues to be an empirical and unpredictable technique. The henil pteros represent an important group of insects that until now have not been effectively controlled by 6-endotox? Many species of plague hernipterans, most notably Lygus species, cause damage to plants and considerable economic losses each year. The hernipteros are among the most economically destructed orders. See Arnett, R.H. 3r. L19853 Ame-i can Insects, / an Nostrand Reihnold Co., Inc., NY). J) e all the hernipteros, the ipdos (Heim tera: Mipdae, including Lygus) are the major pests of crops. c, u food causes damages that weaken the plants, and is a way of transmitting diseases in plants. Adema of L. hesperus, other Lygus plague include: I .. 11 neolari s (Beauv.), L. pratensi (L.), 1. Popp rugulipennis, and cornun green capsid (Lygus pabulmus (L.)). The members of this genre are varied in cotton, potatoes, sugar beets, celery, beans, peaches, apples, alfalfa, pear, plum, quince and various nursery patterns, ornamental plants and vegetable crops. Specific pest mimics include: The potato capsule Ca locoris norvegicus (Grnel.), A pest of potato and cabbage (for example, cabbage, cauliflower *, biocoli, kale, Brussels sprouts, turnip); the bug we made (also called carrot plant bug) ürthops carnpest r i s (L.), a pest of carrots, celery, parsnip, parsley and dill; the apple capsid Plesiocons ru ícolli (Fall.), an apple, currant and graceful blight; the tomato bug Cyrtopeltis rnodestus (Distant), a pest of tomatoes; the sucking fly Cyrto el t i s notatus (Distant), a plague of tobacco, the insect of white marks Spanagonicus albofasciatus (Reuter *), detrimental to grass, which causes particularly noticeable damage to golf pastures; the black acacia bug Diaphnocoris chlorioni (ay), a pest of beets; the bug of the onion plant Labopí dicola aLLii Knight, a pest of wild and cultivated onions; the insect of aLgodon Pseudat ornoscel ís senatus (Reuter), pr * esente in cotton; the bug and ask Adelphoco i s rapius (Say), an occasional pest of cotton and legumes; the four-line Poeci locapsus lineatus (Fabricius) bug, often a pest of garden crops. Other hernipter pests include: Lygaeidae (family of seeds chunche): the bed bug (Blissus leucopterus (Say)) is a pest of corn, sorghums, wheat, me, rice, barley, rye and oats; false bugs (Ny i us spp., for example, N. en cae, N. raphanus lloward) are pests of cabbages, kingdoms, and potatoes. Pentatornidae (family of stinkbugs): the brown stink bug, Euschistus servus (Say), is a cotton pest .. The green stinkbug, Nezara vi riula (I.), Is a widespread pest of seedlings, especially vegetables and legumes; the species of Eury ast er, for * example, II. Austrian (Schr.) and E. integpceps (Put.) (wheat bug, Sunn pest, Senn bug) attack wheat and barley. Coreidae (pumpkin bug family): Anasa ri t i s (DeGreer) (pumpkin bug) is a local pest of pumpkin; A. armí era (Say) (horned pumpkin bug), is an occasional pest of cultivated cucumber. Pyrrhocoridae (family of cotton and red bugs): the cotton ruler D sdercu u ure 11u (Herp eh-Sehaeffer) a flat of < < lgodon "Tingidae (family of The lace bug): Cory rucat (Say) piggy bank is often a plague of roses, maple, apple and chestnut. Other species of Coryt piggy bank cluyen The cotton bug, the chrysanthemum bug, the elm bug and the hawthorn bug. Belostomati ae (family of giant aquatic bugs): it is known that members of this family attack and suck the blood of fish and, therefore, can be a pLaga in fish hatcheries. Members of the Reduvu ae and Cirnicidae (which includes bloodsucking bugs and red bugs, respectively) bite mammals and hunt down diseases that can be contracted by humans. The subject matter of the hemiplous is unusual among insects in various forms: certain digestive enzymes hydrol? T? are absent, such as trypsin; the midgut car-echo of a membrane pep trophi ca, and there is no crop. These characteristics reflect the liquid diet and the suction feeding mode, subject to evolutionary restoring. Due to the differences in diet, mode of feeding, and digestive physiology and biochemistry, it would not necessarily be expected that the proteins that have an insecticidal action against leaf-blasting insects are also effective against hernipids that feed on eggs. Luidos, it was previously found that Bacillus thuri ngiensis PS2U1T6, NRRL B-18750, deposited on January 9, 1991, or a 6-endotoxin thereof, has activity against certain pests. For example, see U.S. Pat. Nos. 5,273,746; 5,298,245 and 5,302,387, which describes the use of PS201T6 of B.t., PS123D1 isolates of B.t. in EP 0409 438. The isolated PS71M3 from B.t. it has been described in EP 0 626 809 and in the patent of E.U.A. No. 5,273,746. The above Patents do not describe or suggest the use of any B isolates. to control hemipteran insect pests. The practice of the present invention provides an alternative to control pests of hemipterans with chemical pesticides, thus allowing a more environmentally friendly insect management and providing a tool for the management of insecticide resistance.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to the use of 6-endotoxmas of Bacillus thupngiensi (B.t.) to control insect pests in ipteros. Specifically exemplified in the present invention is the use of the PS201T6 isolate of B.t. and toxins from it to control the plague of the insect herniptero Lygus he perus. The use of isolates and toxins of B.t "var., Israelensis is also described. the present invention also includes the use of variants of the exemplified isolates and toxins of B.t,. They have substantially the same pesticidal properties as the isolates and toxins exemplified. These variants include mutant microbes. The procedures for obtaining mutants are well known in the microbiological technique. Ultraviolet light and? Nu < Chemical attractants such as nitrosoguanidine are widely used for this purpose.In accordance with the present invention, also recirnant hosts which have been transformed to express Bt toxins can be used. These recolonizing hosts can be, for example. , microorganisms or plants »According to the present invention, hemipteran insect pests can be controlled using the isolate of Bt itself, variants of the isolates of Bt", d ~ endotoxins obtained from said isolates, commercial preparations obtained from cultures of the isolates, or toxins produced by DNA of this isolate In one embodiment, the toxins can be produced by DNA that has been transformed into another host In a disclosed embodiment, the transformed host is a plant.

BRIEF DESCRIPTION OF THE SEQUENCES SEC ID DO NOT. 1 is the nucleotide sequence encoding a PS201T6 toxin of about 30 kDa; TD DE SEC, .. NO. 2 is the deduced amino acid sequence of a PS201T6 toxin of about 30 kDa. SEC ID. NO. 3 is the amino acid sequence of a 20116 toxin runcada of approximately 25 kDa.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the discovery of a new method for controlling * hernipter insect pests which involves contacting said pests with a 6-endotoxin of Bacillus thurmgiensis (B.t.) in a manner in which (Where the herniptero insect ingests the 6-endotoxin.

Specifically exemplified is the use of toxins starting from the B.t isolate. known as PS2Q1? T *. Also exemplified is the use of toxins to isolate from Bac 11 us thup ngiensí var. israelensis (B. t "i.) for center Lar * hernipter pests. Bacillus thrumgiens s isolate PS201T6 has the following characteristics in its biologically pure form and when compared with HD ~ 1 of B.t ": TABLE 1 Comparison of PS201T6 from B.t. and HD-1 of Bt, PS2U1F6 of Bt HD - 1 of Bt Inclusions: the pt pt i co bi pi rain L dal and Pipi rami L moLocular Weight 133,000 130,000 at r of each of the 31,000 68,000 Pr-oteinas déte rrni swimming through SOS-PAGE Variety of guests Cockroaches l pi doptors Dipt o os Worm of the root of aL.

The variety i sraelensis of B. it is well known and is easily recognized by those skilled in the art. The general characteristics associated with the category i sraelensí s of B "< include diptych activity, type H14, and a protein pattern that includes approximately 20 to 33 kD pro duct and, generally, additional proteins of approximately 70 kT) and 130 kT). Other varieties of B.t may also be used. expressing type i sraelensi toxins in accordance with the present invention. These toxins would have a hand similar to the toxins produced by. t .. i. and a similar activity profile, including dipteran activity. It has been reported, by * example, that the rot ipo Ha, 8 b of B.t. go \ ~. mo rr * isoni expresses type B toxins. t. i .. An example of said isolate is PS71I13. As used in the present term, the term "Bacillus turingian islanders", it includes toxins that are common or are related to toxins expressed by B "t" i. , but which are expressed by a variety di tm a B.t ,. In accordance with the present invention, the isolate designated as PS123D1 is specifically exemplified as an isolate of B. t. i. Useful. Of particular interest to control hernipteroid pests are toxins of approximately 28 to 33 kD of a B isolate. t. i. or, preferably, modified forms of this toxin. One embodiment of the present invention includes the use of truncated forms of the 28 kDa PS123D1 toxin to control hernipteros. A specific example is the truncated toxin that has amino acids removed from the N -terminal and is approximately 25 kDa. As described in the present invention, the truncated forms of the toxins can be obtained through the treatment of supernatants from the culture of B. "and / or by developing cultures of B.t. under appropriate conditions to give as a result the production of activated toxins as a result of the advantageous effects of endogenous proteases. Other modifications that cause, for example, solubilization of the B toxin can also be used. t. i. to obtain highly active toxins. Bacillus thuringiensis isolate PS123D1 has the following characteristics in its biologically pure form: l /? TABLE 2 Characteristics of PS123D1 of B.t. PS1 D1 from B.t 1 nclu ions: Amorphic Approximate molecular weight 133, 000 of the proteins by 127, 000 SDS-PAGE 72, 000 28, 000 The crops described *? coughs in this application have been deposited in the Agr *? CuLtural Research Service Patent Ultimate Collection (NRRL), Northern Regional Research Center, 1815 North Umversity Street, Peona, Illinois 61604, USA.

Cultivation Deposit No. Date deposited PS201T6 of Bací 11us thu ri n len i NRRL B-18750 January 9, 1991 NM522 by F. Coll (? MYC2362) NRRL B-21018 December 2, 1992 NM522 of E. Coll (pMYC2357) NRRL B-21017 December 2, L992 PS123D1 of Bacillus thuringiensis NRRL B-21011 October 13, 1992 P 71T13 from Bacillus thuringiensis NRRL B-18930 December 27, 1991 The crops have been deposited under conditions that assure that access to them will be feasible during the suspension of this Patent Application determined by * the Patent and Trademark Commissioner and that is entitled under 37 CFR 1.L4 and 35 U.S.C. 122. Deposits are available as required by foreign patent laws in countries where the counterparts of the Application, or their succession, are filed. However, it must be understood that the availability of the deposits does not constitute an authorization to practice the invention in derogation of the patent rights granted by * governmental action. In addition, the crop deposits will be stored and made available to the public in accordance with the provisions of the 1st rat of Budapest for the Deposit of Microorganisms, that is, they will be stored with all the necessary care to keep them viable and unpolluted for a period of time. period of at least 5 years after the most recent Request for the supply of a sample of a deposit and, in any case, for a period of at least thirty (30) years after the date of deposit. or during the applicable term of any Patent that may require * describe the crops. The depositor appreciates the work to replace the deposits that the deposit must be unable to provide a sample when required, due to the condition of the deposits. All descriptions of the availability of public culture deposits will be irrevocably removed after the granting of a Patent that describes it. In a preferred embodiment of the present invention, a toxin from isolate PS201T6 of B.t is used. to achieve control of hemipteran insect pests. In another preferred embodiment, the PS201T6 toxin is activated. The toxins can be activated, for example, by culturing the microbe, under conditions that facilitate the activation of toxin cUE by the action of compounds that naturally occur or are produced in said culture. Activation can also be achieved by the addition of a compound to a culture of the PS20LT6 strain of Bac 111 us huri ngiens i s or a supernatant thereof., wherein said compound participates in the activation of said toxin by direct action on said toxin or facilitating the action of a second compound. Additional compound FL may be LC), eg, a protease, or a compound that raises the pH of the culture or supernatant. PS201T6 from Bacillus t huringiens is now available to the public, without restriction, by virtue of the enactment of The Patent from EU ,. . Mo. 5,273,746. The plasmid Ib? MYC2362 possesses a gene coding for a 130 kDa PS201T6 toxin. Plasinide? MY02357 possesses a gene encoding a 30 kb PS201I toxin a. Further with respect to examples of gene cloning from PS20LI can be found in EP-A-0 8,830 and UO 95/02694. Other examples of cloning genes from PS71M3 are described in UO 95/02694 and EP-A-Q 57 498.

Genes and toxins. In an embodiment of the present invention, genes are used that encode active toxins of B.t. against insect hernipter pests to transient an appropriate host, genes and toxins useful in accordance with the present invention include not only the full length sequences, but also fragments of these sequences, variants, mutants and fusion proteins that retain the Pesticide activity characteristic of specifically exemplified toxins. As used in the present invention, the terms "variants" or "variations" of genes refer to sequences of nucleotides that encode the same toxins or that encode equivalent toxins that have similar pesticidal activity. As used in the present invention, the term "equivalent toxins" refers to toxins having the same or essentially the same biological activity against the target pests as the toxins described. It would be apparent to one skilled in the art, who has the benefit of this disclosure, that genes encoding toxins active against henipterans can be identified and obtained through various means. The genes can be obtained from the isolate deposited in a culture tank as described above. These genes or portions or vanishing thereof can also be constructed synthetically, for example, by the use of a gene synthesizer. Gene variations can be easily constructed using standard techniques to obtain point mutations. Asunismo, fragments of these genes can be obtained using exonuclease or endonucleases existing in commerce in accordance with patron procedures. For example, they can 10 Enzymes such as Bal 31 or inutagenesis of the site are used to systematically cut nuclei from the ends of the genes. Also, genes encoding active fragments can be obtained using a variety of restriction enzymes. Proteases can be used to directly obtain active fragments of these toxins. Equivalent toxins and / or genes encoding these equivalent toxins can be derived from isolates of B.t. and / or DNA libraries using the teachings provided in the present invention. There are vain methods for obtaining * the pesticide toxins of the present invention. For example, antibodies to the pesticidal toxins described and rei indicated in the present invention can be used to identify and isolate * other toxins from a mixture (ie proteins). Specifically, antibodies can be produced for portions of the toxins that are constants and more distinct from other Bt toxins. These antibodies can then be used to specifically identify equivalent toxins with the characteristic activity by immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) or Uestern blotting, Antibodies pair The toxins described herein invention, or for equivalent toxins, or fragments of these toxins, can be easily prepared using methods for this technique.The genes coding for these toxins can then be obtained from the microorganism.

The fragments and equivalents which retain the pesticidal activity of the exemplified toxins would be within the scope of the present invention. Asunisrno, due to the redundancy of the genetic code, several different sequences (DNA can encode the amino acid sequences described in the present invention) It is well within the aptitude of a person skilled in the art to create * these alternative DNA sequences that encode the same, or essentially the same, toxins »These variant DNA sequences are within the scope of the present invention. As used in the present invention, the reference to "essentially the same" sequence refers to sequences that have substitutions, deletions, additions or insertions of amino acids that do not materially affect the pesticide activity of the pro-inna. Additional method to identify the toxins and genes of the invention is by the use of oligonucleotide probes. These probes are nucleotide sequences that have detection means. As is well known in the art, if the probe molecule and the nucleic acid sample hybridize to form a strong bond between the two molecules, it can reasonably be assumed that the probe and the sample have substantial homology. The detection means provide the means to determine in a known manner whether hybridization has occurred, such probe analysis provides a rapid method to identify the genes encoding the toxin of the present invention. These sequences can be synthesized by the use of a DNA probe using standard procedures, and these nucleotide sequences can also be used as PCR primers to amplify DNA sequences. genes of the present invention.

Recombinant guests. The genes coding for the toxin contained in the isolates described in the present invention can be introduced into a wide variety of microbes or host plants. The Lon expression of the gene for the toxin results in, directly or indirectly, the production of the toxin. With suitable microbial hosts, for example, P udomonas, the microbes can be applied to the site of the pest, where they will proliferate and will be ingested by the pest, resulting in the control of the pest. Alternate ivamen e, the microbe that provides the gene for the toxin can be treated under conditions that prolong the activity of the toxin and stabilize the colula. The treated cell, which retains the toxic activity, can then be applied to the environment of the target pest. Methods of treating microbial cells are described in the Patent of F.U.A. Nos. 4,695,455 and 4,695,462, which is incorporated herein by reference. There is a wide variety of ways to introduce a B.t gene. that encodes a toxin in a host microorganism under conditions that allow stable maintenance and expression of the gene. These methods are well known to those skilled in the art and are described, for example, in the U.S. Patent. No. 5,135,867, which is incorporated herein by reference. In addition, materials and methods for introduci * of Ti., In pLantas to confer on these plants the ability to produce insecticidal toxins, are well known in the art. In a preferred embodiment, the B. le, genes are modified to facilitate optimal stability and expression in the selected host cell. In this regard, the Patent of E.U.A. No. 5,380,831 is incorporated in the present invention as re feroncy.

Mutants Mutants of the isolate described in the present invention can be obtained by methods well known in the art. For example, a sporogen mutant can be obtained by rnutagenesis of an isolate with ethyl-methane sulfonate (EMS). Mutants can be obtained using ultraviolet light and nitrosoguanidy by methods well known in the art. Following are examples illustrating methods for practicing the present invention. These examples should not be considered as limiting. All percentages are by weight and all proportions of the solvent mixture are in volume, unless otherwise indicated. EXAMPLE 1 Culture of the strain PS201T6 of B.t.

A subculture of strain PS201 6 of the isolate of B.t. to inoculate the following peptone medium, g Lucrosa and salts.

Bacto Peptone 7.5 g / 1 Glucose LU g / l KH2PO4 3.4 g / 1 K2HPO4 4.35 g / 1 Saline solution 5.0? Nl / 1 CaCl2 solution 5.0 rnL / 1 Salt solution (100 ml) MgSO4 -7H20 2.46 g MnSO * - H20 0.04 g ZnSOí; -7H20 0.28 g CaCl2 solution (100 ml) CaCl2"2H2? 3.66 g pH 7.2 The solution of salts and the solution of CaCl2 are sterilized with a filter and added to be subjected to autoclaving and to the broth cooked at the time of the mixing. The flasks are incubated at 30 ° C on a rotary shaker at 200 rprn for 64 hours. The above procedure can be easily carried to large fermentors by well-known procedures in the operation. The spores and crystals of B.t. obtained in the above fermentation * can be isolated by procedures well known in the art. A frequently used method is to subject the harvested fermentation broth to separation techniques, for example, centrifugation.

EXAMPLE 2 Production of activated 2D1T6 toxin (201T6-D) Activated 201T6 toxin can be produced by several methods that result in the run of 201T6 toxin. In this regard, reference may be made to UO95 / 02693. In this method, the PS201T6 cultures were harvested by centrifugation and resuspended up to 1/9 part to 1/25 of their original culture volume in 0.1 M Na2 + 3 / NaHC 3., pH 11.0, containing 0.5 rng / rnl of pronase E (Sigma Chemical Company, bacterial protease P-5147 ipo XTV to part of Streptornyces griseus). The suspension was incubated at 37 ° C overnight with mixing. The suspensions were dialyzed against two changes of 50 to 100 volumes each of distilled water or Na2C3 to 0.1M / NaHC3, pH 9.5, to produce * "diallyl suspensions". The suspension resulting from dialysis in Na2CÜ3 a 0. L M / NaHC? 3, pH 9.5, was centrifuged to remove cells, spores and debris. Additional purification from spores and debris can be achieved by filtration through a filter of my Uhatrnan glass fibers, a filter (0.8 micron cellulose acetate, and a cellulose acetate filter). of 0.2 microns to produce * a "filtered supernatant." Dry preparations of the toxin processed before or after filtration were prepared by dialyzing against two changes of 50 to 150 volumes of distilled water, followed by freeze-drying ( lifilized toxin treated with pronase).

EXAMPLE 3 Alternative method to produce activated 201T6 toxin L5 Cultures were harvested (Je PS201T6 by centrifugation and resuspended up to 1/9 part at L / 25 part of their original culture volume in Na2C? 3 at 0.1 M / NaHC? 3, 2-mercaptoethanol at 0.5%, pH 11.0. The suspension is incubated during Approximately 2 hours at room temperature. The suspension was centrifuged to remove cells, spores and debris. Additional purification of spores and debris can be achieved by filtering through a Uhatrnan glass fiber filter, 0.8 cellulose acetate filter, and a 0.2 micron cellulose acetate filter to produce a "filtered supernatant". The suspensions were dialyzed against two changes of 50 to 100 volumes each of distilled water or Na2 CO3 at 0.1 M / NaHC3, pH 9.5, to produce "dial-on suspensions". Dry preparations of the processed toxin were prepared before or after filtration by dialing against two changes of 50 to 100 volumes of distilled water, followed by * lyophilization, the material prepared according to this procedure is referred to in the present invention. invention as 201T6-D.

EXAMPLE 4 Activity of isolates of B.t. Against the insect hept Lygus hesperus Five newly emerged adults (_ <1 week) of I .. hosperus were placed in a plastic cup of l.U ounces for portions (Fabpkal Corp.) with two tiny holes on the side to allow the exchange of gases. A small piece of Scott's MTCROUIPES paper was placed in the bottom of the cup to absorb liquid excreta. Instead of a lid, two pieces of PARAFILM were stretched across the top, sandwiching 200 μL of test solution between them. Culture broth of PS201 6 cells of B.t. "autoprocessed" (activated by resident proteases as described in Example 3), was exposed to adults of L. hesperus. It was estimated that the self-processed broth contains approximately 4.5 rng / rnl of toxin. 1 ml of the whole was diluted to 100 rnl with a 15% sucrose solution. 200 JJI of the 45 μg / rnl solution was used as the test solution. e of or that the insects were fed during 3 days. Mortality after 3 days was compared with that of starving insects and control insects provided as a "target" of 15% sucrose solution that did not contain toxin. After establishing * The activity of 45 μg / rnL of PS201T6 of B.t. after 3 days, a subsequent experiment was carried out to establish the activity of 10 μg / rnL after two days. The representative results are shown in tables 3 and 4.

TABLE 3 Mortality of Lygus hesperus observed in three days and provided by 45 μg of PS201T6 of B.t./mL Percentage Number of Treatment Number of insect insects bite dead insects after cough after live 3 days 3 days Sucrose solution at 15% 25 7 28 45 μg of PS201T6 from B. t./rnl of saccharose solution to 15% 25 17 68 Pnvados of food and water 25 25 100 TABLE 4 Mortality of Lygus hesperus observed in two days and provided by 4.5 μg of PS201T6 of B.t./mL Number of Percentage of Treatment Number of insect insects initially bites dead insects after coughing after live 2 days 2 days Sucrose solution at 15% 50 29 58 45 μg of PS201T6 of B.t./ l of sucrose solution at 15% 50 41 82 Deprived of food and water 50 50 100 EXAMPLE 5 Activity of the isolated PS201T6 of B.t. Against the insect hemipteran Lygus lineolaris As an alternative to the bio-test method on filter paper, an insect is exposed to "SCOÍCH BRITE", or a similar material saturated with the test mixture. The mesh material "SCOTCH 8RITE" is cut from cell fiber bearings "MTNITRANS-BLOT" from BioRad (8 x ÍL crn) in cubes of approximately 0.5 c per side. The cubes are then saturated with test solution and an individual cube is placed in a souffle cup (1.25 oz. Plastic "SOLO" from Cup Company 20 together with an adult of I., 1 meol ar? s "kl container is sealed with a lid for glass of sou fle, and several tiny holes are made in the cup or lid to provide an adequate exchange of air. The insect is allowed to feed at will, and the mortality is measured after four days. Test solutions were prepared by means of samples or blanks from the rotary table for feeding with sterile, filtered (0.2 μrn) saccharose solution in amounts necessary to obtain the desired protein concentrations and LC) 10% sucrose in the final test solution. The proteins can be obtained as described in Example 3. Before sterilizing and filtering the sucrose solution, a green vegetable dye (Iris So.) Was added to provide a visual stimulus to favor feeding. The power supply is L5 I observe easily; During some minutes of exposure to a bucket containing dry test solution, insects could be seen inserting their stylus into the bucket mesh. Shortly afterwards, dry excreta were observed, generally after a few minutes of the beginning of feeding. The results are shown in the Quad-o b. The dose is expressed as μg of toxin / rnL of diet. White control of pH regulator and a BSA control are used as negative controls. The target of the pH regulator was at a concentration equivalent to the bottom in the treatment of 1000 μg / mL. Fl BSA control is done at 500 μg of BSA / rnL of diet., TABLE 5 Summary of mortality data (including total sum through the pH regulator and the negative BSA controls) Number of insects Percentage of diets killed / killed or dead insects 0 (cont'd roles! 21/240 9 10 500 60 / 119 50 1UÜÜ 62/90 69 L5 EXAMPLE 6 Activated toxins from B.t. i, The removal of an N-terminal portion of the 28 kDa 123D1 toxin results in an advantageous activation of this toxin which increases the potency of its activity. The removal of the anuioacids can be achieved by treatment with trypsin or another suitable enzyme, or mixtures of enzymes, such as pronase or endogenous proteins in culture broths of B.t. Other biologically active fragments can be obtained by those skilled in the art using the teachings provided in the present invention. As experts in the technique that have the benefit of this description would easily recognize, the specific means used to develop the cultivation of. t "can be modified to achieve optimal activation of the B toxin. For example, cell density can be modulated by adjusting or changing the culture medium. Likewise, means that have proteases can be used to increase the activation of B toxins. " EXAMPLE 7 Insertion of genes for toxin in plants One aspect of the present invention is the transformation of plants with genes encoding an active toxin cont to heinius pests. The transformed plants are resistant to attack by heteros. The genes encoding pesticidal toxins, as described in the present invention, can be inserted into plant cells using techniques that are well known in the art. For example, there is a large number of cloning vectors comprising a replication system in E. coll and a marker that allows the selection of transfected cells to prepare the insertion of foreign genes into higher plants. The vectors comprise, for example, PBR322, pUC, series M13? np, series pACYC184, etc. Therefore, the sequence encoding the toxin of B.t. it can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation in E. coli. The cells of F. coli are grown in a suitable riutptivo medium, and then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electron oforesis and other biochemical-biomolecular methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cut and an L sequence to the next DNA sequence. Each plasmid sequence can be cloned in the same plasmid or other plasmids. Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the formation of the plant cell, then at least the right border, but often the right edge and the left edge of the T-DNA of the Ti or Ri plasmid. , has to join as the flanking region of the genes that are going to be inserted. The use of T-DNA for the transformation of plant cells has been intensively researched and sufficiently described in EP 120 516; Hoekerna (1985) in: The Bi na ry Plant Vector * System, Of fset-durkke i j Kanters V.B., ALblasserdarn, chapter 5; Fraley et al., Cpt. Rev "Plant Sci. 4: 1-46; and An et al. (1985) EMBO J. 4: 277-287. After the inserted DNA has been integrated into the genome, it is relatively stable there and, as a rule, does not go away again. It normally contains a selection marker * that confers on the transformed plant cells resistance to a biocide or an antibiotic such as kanarnicma, G 418, bleomycin, hygromycin or chloranfeni col, among others. Therefore, the marker * used individually should allow the selection of the cells that have been synthesized more than cells that do not contain the inserted ID1. There are a large number of techniques for inserting DNA into a host plant cell. These techniques include transformation with T-DNA using A g ro c te r i uin e fac e n e s Agrobacterium rhizogenes as an agent of transformation, fusion, injection, biolysis (bombardment of my cells), or electroporation, as well as other possible methods. If agrohazards are used for the transformation, the DNA to be inserted has to be cloned into p-species Les spice, namely in an intermediate vector or in a binary vector. Intermediate vectors can be integrated in the Ti or Ri plasmid by homologous recombination thanks to sequences that are homologous to the T-DNA DNA sequences. The Ti or Ri plasmid also comprises the vir * region necessary for T-DNA transference. The intermediate vectors can not replicate themselves in agrobactepas. The intermediate vector can be transferred in A g ra b te r * i urn t urne fa s s by means of an auxiliary pl (a conjugation) Binary vectors can be replicated by themselves in both E. coli and agrohacteas . They comprise a marker gene for selection and a linker or polylayer that are framed by the right and left border regions of the T-DNA. Puden transforms r-se directly into agrobactepas (Holstors and others [19781 Mol. Gen. Genet, 163: 181-187). The Abacterium used as a host cell comprises a plasmid that has a vir region. The region vires necessary for the transfer of T-DNA in the plant cell. Additional T-DNA content may be present. The bacterium thus transformed is used for the trans formation of plant cells. Plant explants can be advantageously grown with Agrobact erium turnefaciens or Agrobacterium rhi ogenes for the transfer of DNA into the plant cell.

Replenished plants may then be regenerated from the infected plant material (eg, pieces of leaves, stem segments, roots, but also protoplasts or cells grown in suspension) in a suitable medium, which may contain antibiotics or biocides for selection. The plants thus obtained can then be tested for the presence of the inserted DNA No special demand is made for the plasmids in the case of injection and elect roporation It is possible to use ordinary forms such as, for example, pUC derivatives. The transformed cells grow inside the plants in the usual way, they can form germ cells and transmit the transformed characteristic (s) to the plants of the progeny, these plants can be cultivated in the normal way and crossing with plants that have the same factor-is hereditary transformed or other factor-is hereditary.The resulting hybrid individuals have the properties corresponding genotypes. In a preferred embodiment, the plants will be transformed with genes where codon usage has been optimized for plants, see, for example, US Patent No. 5,380,831. plants that encode a truncated toxin The runna toxin will typically encode about 55% to about 80% of the total length of the toxin Methods for creating * synthetic Bt genes for use in plants are known in the art.

EXAMPLE 8 Cloning of novel B.t. genes in insect viruses Vain viruses are known to infect insects. These viruses include, for example, baculoví rus and entomopoxvi rus. In one embodiment of the present invention, genes active against hernipterans, as described herein, can be placed within the genome of the insect virus, thereby increasing the pathogenesis of the virus. Methods for constructing insect viruses comprising genes for B.t toxin. they are well known and easily practiced by those skilled in the art. These procedures are described, for example, in Merryweather et al. (Merryweather, AT, U. Ueyer, MPG Harris, M. Hirst, T. Booth, RD Possee [1990] 3. Gen. Vi.R.71: 1535-1544) and Martens and others (Martens, JWM, G. Honee, D. Zuide, JUM van Lent, ß. Visser, J.M. Vlak CL990T Appl. Env i ronrnental Microbiol., _56 (9) 12764-2770). It is to be understood that the examples and embodiments described in the present invention are for illustrative purposes only and that various modifications or changes in light thereof will be suggested by the experts in the art and should be included within the spirit and competence of this art. application and the scope of the attached indications.

LIST OF SEQUENCES NEORM CI ON GENER L: (l) INFORMATION OF THE APPLICANT NAME OF THE SUN rOJTAN "ÍT: MYCOGEN CORPORA1101 ADDRESS: 5501 Oberl in dn ve CITY: San diego ESTADO / PROVTNC IA: Cali orni COUNTRY: EU., POSTAL CODE: 92121 TELEPHONE: C 61 q) 453 8030 TELEFAX: (619) 3-69 1 (II) TITLE OF I A RNVFIMOION: CONTROVEMENT OF INSECTS PESTS HEM1PTFROS WITH PAC TU US THURING I FNSTS (III) NUMBER J) F SEQUENCES: 3 (iv) ADDRESS FOR CORRESPONDENCE- (A) RECIPIENT: Sali anchik & Saliwanchik (B) STREET: 2421 N.W. 41ST Street, Suite AL (C) CITY: Gainesville (D) STATE: FL (E) COUNTRY: UNITED STATES OF NORTH AMERICA RTCA (P) POSTAL CODE: 32606 (v) LEGIBLE COMPUTER FORM: (A) TYPE OF MEANS: Disco Flexible (O) COMPUTER: Corn? < * .t ibl e WITH IBM PC (C) OPERATIONAL STSTFMA: PC-DOC / MS-DOS (D) PROGRAMS: Patentin Relay # 1.0, Version # 1.25 (vi) COMMON DATA OF THE APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: (C) CLASSIFICATION: (vile) PRE-REQUIRED DATA OF THE APPLICATION: (A) APPLICATION NUMBER: US 08 / 475,924 (D) DATE OF SUBMISSION: JUNE 07 Dt 1995 (C) CLASSIFICATION: (vm) EMPLOYEE / AGENT INFORMATION: (A) NAME: Sanders, Jay M. (B) REGISTRY NUMBER: 39,355 (C) REFERENCE / CASE NUMBER: MA96. Cl (IX) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 352-375-8100 (B) TELEFAX: 352-372-5800 (2) INFORMATION FOR SEC ID. NO: l: (i) CHARACTERISTICS DF THE SEQUENCE: (A) LENGTH: '95 stops (Je bases (B) TYPE: nucLeic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear (II) MOLECULE TTPO : DNA (genon) (III) HYPOTHETIC: No (iv) ANTI ENTDO: No (vi) SOURCE ORTGINAI: (A) ORGANISM: Bacillus t hurí ngi ensí s (B) CFPA: neoleoensis (C) INDIVIDUAL AISI ADO: PS201T6 ( vi l) SOURCE I? MFD1ATA: (A) GENOTECA: Genn LarnabdaGern (TM) -ll by Kenneth E. Narva (B) CLON: 201T535 (i) DESCRIPTION OF THE SECUENCTA: SEC ID. DO NOT. L: ATGAAAGAGT «= AATTTACTA CAATGAAGAA? ATGAAATAC AAATTTCACA A-3GAAACTGT 60 TTCCCAC? AAG AATTAGGACA TAATCCTTGG AGACAACCTC AATCCA-CAGC AAGAGTTATT 120 TATTTAAAAG TAAAAGATCC TATTC? ATACT ACTCAATTAT TAGAAATAAC AGAAATCGAA 180 A? TCCCAATT ATGTATTACA AGCTATTCAA C? A «3CTGCTG CCTTCCAAGA TGCATTAGTA 240 CCAACT «3AAA C? GAATTTGG ACSAAGCCATT AGATTTAGTA TGCCTAAAGG ATTAGAAGTT 300 GCAAAAACTA TTCAACCTAA GGGTGCTGTT GTTGCTTACA CAGATCAAAC TCTGTCACAA 360 AßCAA «= AACC AAGTTAGTGT TATGATTGAT AGAGTTATTA GTGTTTTAAA AACTGTAATG 420 GGAGTAGCTC TTAGTGGTTC CATTATAACT CAATTAACAG CTGCTATCAC TGATACTTTT 480 ACAAACCTTA ATACACAAAA AGATTCTGCT T «3GGTTTTTT GGGGAAAAGA AACTTCACAT 540 CAAACAAATT ACAC "ATATAA TGT" CATGTTT GCAATTCAAA ATGAAACAAC TGGACGCGTA 600? TGATGTGTG TACCTATTGG ATTTGAAATT AGAGTATTTA CTGATAAAAG AACAGTTTTA 660 TTTTTAACAA CTAAAGATTA CGCTAATTAT AGTGTGAATA TTCAAACCCT AAGGTTTGCT 720 CAACCACTTA TTGATAGCAG AGCACTTTCA ATTAATGATT TATCAGAAGC ACTTACSATCT 780 TCTAAATATT TATAC 795 (2) INFORMATION FOR SEC ID. NO: 2: (I) CHARACTERISTICS DF THE SEQUENCE: (A) LENGTH: 265 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: i dividual (ü) TOPOLOGY: linear (II) UPO DF MOLECULA: prot e would go (lll) HYPOTHETIC: Sl (IV) ANTI ENRIET: o (Vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus t hurí ngiensis (B) CEPA: neoleoensis (C) INDIVIDUAL ISOLATED: PS201T6 (vile) IMMEDIATE SOURCE: (A) ) GENOTECA: Library LarnbdaGem (I) -ll by Kenneth E. Na r () CLON: 201T635 (Xl) DESCRIPTION OF THE SEQUENCE: ID DF SEC. DO NOT. 2: Mßt Lys Glu be He Tyr Tyr Asn Glu Glu Asn Glu llß Gln lie be 1 5 10 15 Gln Gly Asn cys Phe Pro Glu Glu Leu Gly His Asn Pro Trp Arg Gln 20 25 30 Pro Gln be Thr Ala Arg Val He Tyr Leu Lys to Lys Asp Pro llß 35 40 45 Asp Thr Thr Gln Leu Leu Glu He Thr Glu He Glu Asn Pro Asn Tyr 50 55 60 Val Leu Gn Ala He Gln Leu Ala Ala Ala Phe Gln Asp Ala Leu Val 65 70 75 80 Pro Thr Glu Thr Glu Phß Gly Glu Wing He Arg Phe be Mßt Pro Lys 85 90 35 Gly Leu Glu Val Wing Lys Thr He Gln Pro Lys Gly Wing Val Val Wing 100 105 110 Tyr Thr Asp Gln Thr Leu be Gln be Asn Asn Gln Val be Val Met 115 120 125 llß Asp Arg Val He Ser Val Leu Lys Thr Val Met Gly Val Ala Leu 130 135 140 Ser Gly be He He Thr Glrn Leu Thr Ala Wing Thr Asp Thr Phe 145 150 155 160 t & n Leu Asn Thr Gln Lys Asp Ser Wing Trp Val Phß Trp Gly Lyß 165 170 175 Glu Thr be Bis Gln Thr Asn Tyr Thr Tyr Asn Val Mßt Phß Ala He 180 185 190 ß? N Asn He Gly Phe Glu lie Arg val Phe Thr Asp Lys Arg Thr Val Leu Phe Leu Thr Thr 210 215 220 Lys Aso Tyr Ala Asn Tyr be Val Asn He ßln Thr Leu Arg Phe Wing 225"230 235 240 Gin P-o Leu He Asp Ser Arg Ala Leu be He Asn Asp Leu Ser Glu 245 250 255 Wing Leu Arg be Lys Tyr Leu Tyr 260 265 (2) INFORMATION FOR SEC ID. NO: 3: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 222 amino acids (B) TYPE: amino acid (C) CHAIN UPO: individual (D) TOPOLOGY: linear (11) TYPE OF MOLECULE: proLe ma (111) ) HYPOTHETIC: Sl (ív) ANTISENT TDO _ No (vi) SOURCE ORIGINAI: (A) ORGANISM: Bacillus t hu mgiensí s (B) CEPA: neoleoensis (C) ISOLATED riMÜIVIDUAL: PS201T6 (xi) DESCRTPCION OF THE SEQUENCE: ID OF SEC. DO NOT. 3rd Val Lys Asp pro As As Thr Thr Gln Leu Leu Glu He Thr Glu ll 1 5 10 15 Glu Asn Pro Asn Tyr val Leu Gln? La ll? Gln Leu Ala Wing Ala Ph? 20 25 30 Gln Asp Ala Leu at Pro Thr Glu Thr Glu Phß Gly Glu Ala llß Arg 35 40 45 Phß be Met Pro Lys Gly Leu Glu Val Wing Lys Thr llβ Gln Pro Lys 50 55 60 Gly Wing Val to Wing Tyr Thr Asp Gln Thr Leu Ser Gln Ser Asn? Sn 65 70 75 80 Gln to be Val Met He Asp Arg Val To be to Leu Lys Thr Val 85 90 95 Met Gly to Ala Leu be Gly be He He Thr Gln Leu Thr Ala Ala 100 105 110 ID "fi ot < - * -" Be * ^ "D ^ * a < ™? W- ae • *" - * or * > * E cn m "* - ^ -» * t ** e »« - «^ ^^«. Í ^. T ** - *** 115 120 125 Val Phe Trp Gly Lys Glu Thr Ser His Gln Thr Asn Tyr Thr Tyr Asn 130 135 140 Val Met Phe Ala He Gln Asn Glu Thr Thr Gly Arg Val Met Met Cys 145 150 155 160 Val Pro He Gly Phe Glu He Arg Val Phe Thr Asp Lys Arg Thr Val 165 170 175 Leu Phe Leu Thr Thr Lys Asp Tyr Wing Asn Tyr Ser Val Asn He Gln 180 185 190 Thr Leu Arg Phe Wing n Pro Leu He Asp Be Arg Ala Leu be He 195 200 205 Asn Asp Leu Ser Glu Ala Leu Arg be Ser Lys Tyr Leu Tyr 210 215 220

Claims (6)

NOVELTY OF THE INVENTION CLAIMS

1. A network for controlling herbal insect pests that includes administering to these pests, or to the environment of said pests, an effective amount of a 6-endotoxin of Bacillus thunngiensis (Bt) to control hemipteran insect pests. .

2. The method according to claim 1, which comprises contacting said insect pest with a 6-endotoxin from a Bací 11 us thur * isolate? ngiensis goes r. Israelensis

3. The method according to claim 1, which comprises contacting said insect hernipter pests with a d-endotoxin from a side of a Bacillus thunngiensis selected from the group consisting of PS201T6, PS123D1, PS71M3, and (ilutants thereof)

4. The method according to claim 1, wherein the d-endotoxin of Bacillus thuri ngiensi s is a toxin of Total length, or an active fragment against herniptes, of the same

5. The method according to claim 3, wherein said toxin of Bacillus thupngiensis is an activated toxin 6.- The method according to the rei indication 4, wherein said activation is offended by truncation of said toxin from the ro to PS201 I? * of Bac 111 us t hurí ng onsí s "7.- The method according to La rei indication 1, wherein said toxin The method of conformity with claim 1, wherein said plague of hernipteros is L and us hesperus 9. The method according to claim 1, wherein said method is based on PS201T

6. plague of hemip + ros is L gus 1 ineolari 10. Ll m all of according to claim 1, wherein d-endotoxin (Je Bací 11 us t hu ingiensis is administered to pests and hepuptorous insects on plants by inserting an insecticidal structural gene of Bt "into the genome of the plant, so that the 6-endotoxin of B. it is expressed in the tissue of the plant that the herbal insect pest swallows. The method according to claim LO, wherein said gene of B.t. encodes a PS201T6 toxin or a fragment or vanant active against heimpteros thereof.